Processes for preparing phosphorodiamidate morpholino oligomers

ABSTRACT

Provided herein are processes for preparing an oligomer (e.g., a morpholino oligomer). The synthetic processes described herein may be advantageous to scaling up oligomer synthesis while maintaining overall yield and purity of a synthesized oligomer.

RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/508,256, filed May 18, 2017, U.S. ProvisionalPatent Application Ser. No. 62/341,049, filed May 24, 2016, U.S.Provisional Patent Application Ser. No. 62/340,953, filed May 24, 2016,U.S. Provisional Patent Application Ser. No. 62/357,134, filed Jun. 30,2016, and U.S. Provisional Patent Application Ser. No. 62/357,166, filedJun. 30, 2016. The entire contents of the above-referenced provisionalpatent applications are incorporated herein by reference.

BACKGROUND

Antisense technology provides a means for modulating the expression ofone or more specific gene products, including alternative spliceproducts, and is uniquely useful in a number of therapeutic, diagnostic,and research applications. The principle behind antisense technology isthat an antisense compound, e.g., an oligonucleotide, which hybridizesto a target nucleic acid, modulates gene expression activities such astranscription, splicing or translation through any one of a number ofantisense mechanisms. The sequence specificity of antisense compoundsmakes them attractive as tools for target validation and genefunctionalization, as well as therapeutics to selectively modulate theexpression of genes involved in disease.

Duchenne muscular dystrophy (DMD) is caused by a defect in theexpression of the protein dystrophin. The gene encoding the proteincontains 79 exons spread out over more than 2 million nucleotides ofDNA. Any exonic mutation that changes the reading frame of the exon, orintroduces a stop codon, or is characterized by removal of an entire outof frame exon or exons, or duplications of one or more exons, has thepotential to disrupt production of functional dystrophin, resulting inDMD.

Recent clinical trials testing the safety and efficacy of spliceswitching oligonucleotides (SSOs) for the treatment of DMD are based onSSO technology to induce alternative splicing of pre-mRNAs by stericblockade of the spliceosome (Cirak et al., 2011; Goemans et al., 2011;Kinali et al., 2009; van Deutekom et al., 2007). However, despite thesesuccesses, the pharmacological options available for treating DMD arelimited.

Golodirsen is a phosphorodiamidate morpholino oligomer (PMO) designed toskip exon 53 of the human dystrophin gene in patients with DMD who areamendable to exon 53 skipping to restore the read frame and produce afunctional shorter form of the dystrophin protein.

Although significant progress has been made in the field of antisensetechnology, there remains a need in the art for methods of preparingphosphorodiamidate morpholino oligomers with improved antisense orantigene performance.

SUMMARY

Provided herein are processes for preparing phosphorodiamidatemorpholino oligomers (PMOs). The synthetic processes described hereinallow for a scaled-up PMO synthesis while maintaining overall yield andpurity of a synthesized PMO.

Accordingly, in one aspect, provided herein is a process for preparingan oligomeric compound of Formula (A):

In certain embodiments, provided herein is a process for preparing anoligomeric compound of Formula (G):

In yet another embodiment, the oligomeric compound of the disclosureincluding, for example, some embodiments of an oligomeric compound ofFormula (G), is an oligomeric compound of Formula (XII):

For clarity, the structural formulas including, for example, oligomericcompound of Formula (C) and Golodirsen depicted by Formula (XII), are acontinuous structural formula from 5′ to 3′, and, for the convenience ofdepicting the entire formula in a compact form in the above structuralformulas, Applicants have included various illustration breaks labeled“BREAK A” and “BREAK B.” As would be understood by the skilled artisan,for example, each indication of “BREAK A” shows a continuation of theillustration of the structural formula at these points. The skilledartisan understands that the same is true for each instance of “BREAK B”in the structural formulas above including Golodirsen. None of theillustration breaks, however, are intended to indicate, nor would theskilled artisan understand them to mean, an actual discontinuation ofthe structural formulas above including Golodirsen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 and FIG. 2 show a representative analytical high performanceliquid chromatography (HPLC) chromatogram of a synthesized anddeprotected Golodirsen (SRP-4053) crude drug substance (see Example 4).

FIG. 3 and FIG. 4 show a representative analytical HPLC chromatogram ofa purified Golodirsen drug substance solution (see Example 5).

FIG. 5 and FIG. 6 show a representative analytical HPLC chromatogram ofa desalted and lyophilized Golodirsen drug substance (see Example 5).

DETAILED DESCRIPTION

Provided herein are processes for preparing a morpholino oligomer. The amorpholino oligomer described herein displays stronger affinity for DNAand RNA without compromising sequence selectivity, relative to native orunmodified oligonucleotides. In some embodiments, the morpholinooligomer of the disclosure minimizes or prevents cleavage by RNase H. Insome embodiments, the morpholino oligomer of the disclosure does notactivate RNase H.

The processes described herein are advantageous in an industrial-scaleprocess and can be applied to preparing quantities of a morpholinooligomer in high yield and scale (e.g., about 1 kg, about 1-10 kg, about2-10 kg, about 5-20 kg, about 10-20 kg, or about 10-50 kg).

Definitions

Listed below are definitions of various terms used to describe thisdisclosure. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

“Base-protected” or “base protection” refers to protection of thebase-pairing groups, eg purine or pyrimidine bases, on the morpholinosubunits with protecting groups suitable to prevent reaction orinterference of the base-pairing groups during stepwise oligomersynthesis. (An example of a base-protected morpholino subunit is theactivated C subunit Compound (C) having a CBZ protecting group on thecytosine amino group depicted below.)

An “activated phosphoramidate group” is typically achlorophosphoramidate group, having substitution at nitrogen which isdesired in the eventual phosphorodiamidate linkage in the oligomer. Anexample is (dimethylamino)chlorophosphoramidate, i.e. —O—P(═O)(NMe₂)Cl.

The term “support-bound” refers to a chemical entity that is covalentlylinked to a support-medium.

The term “support-medium” refers to any material including, for example,any particle, bead, or surface, upon which an oligomer can be attachedor synthesized upon, or can be modified for attachment or synthesis ofan oligomer. Representative substrates include, but are not limited to,inorganic supports and organic supports such as glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TEFLON, etc.), polysaccharides, nylon ornitrocellulose, ceramics, resins, silica or silica-based materialsincluding silicon and modified silicon, carbon, metals, inorganicglasses, plastics, optical fiber bundles, and a variety of otherpolymers. Particularly useful support-medium and solid surfaces for someembodiments are located within a flow cell apparatus. In someembodiments of the processes described herein, the support-mediumcomprises polystyrene with 1% crosslinked divinylbenzene.

In some embodiments, representative support-medium comprise at least onereactive site for attachment or synthesis of an oligomer. For example,in some embodiments, a support-medium of the disclosure comprises one ormore terminal amino or hydroxyl groups capable of forming a chemicalbond with an incoming subunit or other activated group for attaching orsynthesizing an oligomer.

Some representative support-medium that are amenable to the processesdescribed herein include, but are not limited to, the following:controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g.,Alul, et al., Nucleic Acids Research 1991, 19, 1527); silica-containingparticles, such as porous glass beads and silica gel such as that formedby the reaction of trichloro-[3-(4-chloromethyl)phenyl]propylsilane andporous glass beads (see Parr and Grohmann, Angew. Chem. Internal. Ed.1972, 11, 314, sold under the trademark “PORASIL E” by WatersAssociates, Framingham, Mass., USA); a mono ester of1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, TetrahedronLett., 1970, 4503, sold under the trademark “BIOPAK” by WatersAssociates); TENTAGEL (see, e.g., Wright, et al., Tetrahedron Letters1993, 34, 3373); cross-linked styrene/divinylbenzene copolymer beadedmatrix, or POROS, a copolymer of polystyrene/divinylbenzene (availablefrom Perceptive Biosystems); soluble support-medium such as polyethyleneglycol PEG's (see Bonora et al., Organic Process Research & Development,2000, 4, 225-231); PEPS support, which is a polyethylene (PE) film withpendant long-chain polystyrene (PS) grafts (see Berg, et al., J. Am.Chem. Soc., 1989, 111, 8024 and International Patent Application WO1990/02749); copolymers of dimethylacrylamide cross-linked withN,N′-bisacryloylethylenediamine, including a known amount ofN-tertbutoxycarbonyl-beta-alanyl-N′-acryloylhexamethylenediamine (seeAtherton, et al., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979,8, 351, and J. C. S. Perkin I 538 (1981)); glass particles coated with ahydrophobic cross-linked styrene polymer (see Scott, et al., J. Chrom.Sci., 1971, 9, 577); fluorinated ethylene polymer onto which has beengrafted polystyrene (see Kent and Merrifield, Israel J. Chem. 1978, 17,243 and van Rietschoten in Peptides 1974, Y. Wolman, Ed., Wiley andSons, New York, 1975, pp. 113-116); hydroxypropylacrylate-coatedpolypropylene membranes (Daniels, et al., Tetrahedron Lett. 1989, 4345);acrylic acid-grafted polyethylene-rods (Geysen, et al., Proc. Natl.Acad. Sci. USA, 1984, 81, 3998); a “tea bag” containingtraditionally-used polymer beads (Houghten, Proc. Natl. Acad. Sci. USA,1985, 82, 5131); and combinations thereof.

The term “flow cell apparatus” refers to a chamber comprising a surface(e.g., solid surface) across which one or more fluid reagents (e.g.,liquid or gas) can be flowed.

The term “deblocking agent” refers to a composition (e.g., a solution)comprising a chemical acid or combination of chemical acids for removingprotecting groups. Exemplary chemical acids used in deblocking agentsinclude halogenated acids, e.g., chloroacetic acid, dichloroacetic acid,trichloroacetic acid, fluoroacetic acid, difluoroacetic acid, andtrifluoroacetic acid. In some embodiments, a deblocking agent removesone or more trityl groups from, for example, an oligomer, asupport-bound oligomer, a support-bound subunit, or other protectednitrogen or oxygen moiety.

The terms “halogen” and “halo” refer to an atom selected from the groupconsisting of fluorine, chlorine, bromine, and iodine.

The term “capping agent” refers to a composition (e.g., a solution)comprising an acid anhydride (e.g., benzoic anhydride, acetic anhydride,phenoxyacetic anhydride, and the like) useful for blocking a reactivecite of, for example, a support-medium forming a chemical bond with anincoming subunit or other activated group.

The term “cleavage agent” refers to a composition (e.g., a liquidsolution or gaseous mixture) comprising a chemical base (e.g., ammoniaor 1,8-diazabicycloundec-7-ene) or a combination of chemical basesuseful for cleaving, for example, a support-bound oligomer from asupport-medium.

The term “deprotecting agent” refers to a composition (e.g., a liquidsolution or gaseous mixture) comprising a chemical base (e.g., ammonia,1,8-diazabicycloundec-7-ene or potassium carbonate) or a combination ofchemical bases useful for removing protecting groups. For example, adeprotecting agent, in some embodiments, can remove the base protectionfrom, for example, a morpholino subunit, morpholino subunits of amorpholino oligomer, or support-bound versions thereof.

The term “solvent” refers to a component of a solution or mixture inwhich a solute is dissolved. Solvents may be inorganic or organic (e.g.,acetic acid, acetone, acetonitrile, acetyl acetone, 2-aminoethanol,aniline, anisole, benzene, benzonitrile, benzyl alcohol, 1-butanol,2-butanol, i-butanol, 2-butanone, t-butyl alcohol, carbon disulfide,carbon tetrachloride, chlorobenzene, chloroform, cyclohexane,cyclohexanol, cyclohexanone, di-n-butylphthalate, 1,1-dichloroethane,1,2-dichloroethane, diethylamine, diethylene glycol, diglyme,dimethoxyethane (glyme), N,N-dimethylaniline, dimethylformamide,dimethylphthalate, dimethylsulfoxide, dioxane, ethanol, ether, ethylacetate, ethyl acetoacetate, ethyl benzoate, ethylene glycol, glycerin,heptane, 1-heptanol, hexane, 1-hexanol, methanol, methyl acetate, methylt-butyl ether, methylene chloride, 1-octanol, pentane, 1-pentanol,2-pentanol, 3-pentanol, 2-pentanone, 3-pentanone, 1-propanol,2-propanol, pyridine, tetrahydrofuran, toluene, water, p-xylene).

The phrases “morpholino oligomer” and “phosphorodiamidate morpholinooligomer” or “PMO” refers to an oligomer having morpholino subunitslinked together by phosphorodiamidate linkages, joining the morpholinonitrogen of one subunit to the 5′-exocyclic carbon of an adjacentsubunit. Each morpholino subunit comprises a nucleobase-pairing moietyeffective to bind, by nucleobase-specific hydrogen bonding, to anucleobase in a target.

The term “EG3 tail” refers to triethylene glycol moieties conjugated tothe oligomer, e.g., at its 3′- or 5′-end. For example, in someembodiments, “EG3 tail” conjugated to the 3′ end of an oligomer can beof the structure:

The terms “about” or “approximately” are generally understood by personsknowledgeable in the relevant subject area, but in certain circumstancescan mean within ±10%, or within ±5%, of a given value or range.

Processes for Preparing Morpholino Oligomers

Synthesis is generally prepared, as described herein, on asupport-medium. In general a first synthon (e.g. a monomer, such as amorpholino subunit) is first attached to a support-medium, and theoligomer is then synthesized by sequentially coupling subunits to thesupport-bound synthon. This iterative elongation eventually results in afinal oligomeric compound. Suitable support-media can be soluble orinsoluble, or may possess variable solubility in different solvents toallow the growing support-bound polymer to be either in or out ofsolution as desired. Traditional support-media are for the most partinsoluble and are routinely placed in reaction vessels while reagentsand solvents react with and/or wash the growing chain until the oligomerhas reached the target length, after which it is cleaved from thesupport, and, if necessary, further worked up to produce the finalpolymeric compound.

More recent approaches have introduced soluble supports includingsoluble polymer supports to allow precipitating and dissolving theiteratively synthesized product at desired points in the synthesis(Gravert et al., Chem. Rev., 1997, 97, 489-510).

Provided herein are processes for preparing morpholino oligomers).

Thus, in one aspect, provided herein is a process for preparing acompound of Formula (II):

wherein R¹ is a support-medium;

wherein the process comprises contacting a compound of Formula (A1):

-   -   wherein R¹ is a support-medium and R³ is selected from the group        consisting of trityl, monomethoxytrityl, dimethoxytrityl and        trimethoxytrityl;        with a deblocking agent to form the compound of Formula (II).

In another aspect, provided herein is a process for preparing a compoundof Formula (A3):

-   -   wherein R¹ is a support-medium, and R³ is selected from the        group consisting of trityl, monomethoxytrityl, dimethoxytrityl        and trimethoxytrityl;        wherein the process comprises contacting a compound of Formula        (II):

wherein R¹ is a support-medium;

with a compound of Formula (A2):

-   -   wherein R³ is selected from the group consisting of trityl,        monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;        to form the compound of Formula (A3).

In still another aspect, provided herein is a process for preparing acompound of Formula (IV):

wherein R¹ is a support-medium;

wherein the process comprises contacting a compound of Formula (A3):

-   -   wherein R¹ is a support-medium, and R³ is selected from the        group consisting of trityl, monomethoxytrityl, dimethoxytrityl        and trimethoxytrityl;        with a deblocking agent to form a compound of Formula (IV).

In yet another aspect, provided herein is a process for preparing acompound of Formula (A5):

wherein R¹ is a support-medium, R³ is selected from the group consistingof trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and

R⁴ is selected from the group consisting of:

wherein the process comprises contacting a compound of Formula (IV):

wherein R¹ is a support-medium;

with a compound of Formula (A4):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

to form a compound of Formula (A5).

In another aspect, provided herein is a process for preparing a compoundof Formula (A9):

wherein n is an integer from 10 to 40, R¹ is a support-medium, R³ isselected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

wherein the process comprises the sequential steps of:(a) contacting a compound of Formula (IV):

wherein R¹ is a support-medium;

with a compound of Formula (A4):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

to form a compound of Formula (A5):

wherein R¹ is a support-medium, R³ is selected from the group consistingof trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and

R⁴ is selected from the group consisting of:

and(b) performing n−1 iterations of the sequential steps of:

(b1) contacting the product formed by the immediately prior step with adeblocking agent; and

(b2) contacting the compound formed by the immediately prior step with acompound of Formula (A8):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

to form a compound of Formula (A9).

In yet another aspect, provided herein is a process for preparing acompound of Formula (A10):

wherein n is an integer from 10 to 40, R¹ is a support-medium, and R⁴is, independently for each occurrence, selected from the groupconsisting of:

wherein the process comprises contacting a compound of Formula (A9):

wherein n is an integer from 10 to 40, R¹ is a support-medium, R³ isselected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

and with a deblocking agent to form a compound of Formula (A10).

In still another aspect, provided herein is a process for preparing acompound of Formula (A11):

wherein n is an integer from 10 to 40, and R⁴ is, for each occurrenceindependently selected from the group consisting of:

andwherein the process comprises contacting the compound of Formula (A10):

wherein n is an integer from 10 to 40, R¹ is a support-medium, and R⁴is, independently for each occurrence, selected from the groupconsisting of:

with a cleaving agent to form a compound of Formula (A11).

In another aspect, provided herein is a process for preparing anoligomeric compound of Formula (A):

wherein n is an integer from 10 to 40, and each R² is, independently foreach occurrence, selected from the group consisting of:

wherein the process comprises contacting a compound of Formula (A11):

wherein n is an integer from 10 to 40, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

and with a deprotecting agent to form the oligomeric compound of Formula(A).

In another aspect, provided herein is a process for preparing anoligomeric compound of Formula (A):

wherein n is an integer from 10 to 40, and each R² is, independently foreach occurrence, selected from the group consisting of:

wherein the process comprises the sequential steps of:(a) contacting a compound of Formula (A1):

-   -   wherein R¹ is a support-medium and R³ is selected from the group        consisting of trityl, monomethoxytrityl, dimethoxytrityl and        trimethoxytrityl;        with a deblocking agent to form the compound of Formula (II):

wherein R¹ is a support-medium;

(b) contacting the compound of Formula (II) with a compound of Formula(A2):

-   -   wherein R³ is selected from the group consisting of trityl,        monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;        to form a compound of Formula (A3):

-   -   wherein R¹ is a support-medium, and R³ is selected from the        group consisting of trityl, monomethoxytrityl, dimethoxytrityl        and trimethoxytrityl;        (c) contacting the compound of Formula (A3) with a deblocking        agent to form a compound of Formula (IV):

wherein R¹ is a support-medium;

(d) contacting the compound of Formula (IV) with a compound of Formula(A4):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

to form a compound of Formula (A5):

wherein R¹ is a support-medium, R³ is selected from the group consistingof trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and

R⁴ is selected from the group consisting of:

(e) performing n−1 iterations of the sequential steps of:

(e1) contacting the product formed by the immediately prior step with adeblocking agent; and

(e2) contacting the compound formed by the immediately prior step with acompound of Formula (A8):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ is,independently for each compound of Formula (A8), selected from the groupconsisting of:

to form a compound of Formula (A9):

wherein n is an integer from 10 to 40, R¹ is a support-medium, R³ isselected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

and(f) contacting the compound of Formula (A9) with a deblocking agent toform a compound of Formula (A10):

wherein n is an integer from 10 to 40, R¹ is a support-medium, and R⁴is, independently for each occurrence, selected from the groupconsisting of:

(g) contacting the compound of Formula (A10) with a cleaving agent toform a compound of Formula (A11):

wherein n is an integer from 10 to 40, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

and(h) contacting the compound of Formula (A11) with a deprotecting agentto form the oligomeric compound of Formula (A).

In one embodiment, step (d) or step (e2) further comprises contactingthe compound of Formula (IV) or the compound formed by the immediatelyprior step, respectively, with a capping agent.

In another embodiment, each step is performed in the presence of atleast one solvent.

In yet another embodiment, the deblocking agent used in each step is asolution comprising a halogenated acid.

In still another embodiment, the deblocking agent used in each step iscyanoacetic acid.

In another embodiment, the halogenated acid is selected from the groupconsisting of chloroacetic acid, dichloroacetic acid, trichloroaceticacid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid.

In another embodiment, the halogenated acid is trifluoroacetic acid.

In yet another embodiment, at least one of steps (a), (c), (e), and (g1)further comprise the step of contacting the deblocked compound of eachstep with a neutralization agent.

In still another embodiment, each of steps (a), (c), (e), and (g1)further comprise the step of contacting the deblocked compound of eachstep with a neutralization agent.

In another embodiment, the neutralization agent is in a solutioncomprising dichloromethane and isopropyl alcohol.

In yet another embodiment, the neutralization agent is a monoalkyl,dialkyl, or trialkyl amine.

In still another embodiment, the neutralization agent isN,N-diisopropylethylamine.

In another embodiment, the deblocking agent used in each step is asolution comprising 4-cyanopyridine, dichloromethane, trifluoroaceticacid, trifluoroethanol, and water.

In yet another embodiment, the capping agent is in a solution comprisingethylmorpholine and methylpyrrolidinone.

In still another embodiment, the capping agent is an acid anhydride.

In another embodiment, the acid anhydride is benzoic anhydride.

In another embodiment, the compounds of Formula (A4) and Formula (A8)are each, independently, in a solution comprising ethylmorpholine anddimethylimidazolidinone.

In another embodiment, the cleavage agent comprises dithiothreitol and1,8-diazabicyclo[5.4.0]undec-7-ene.

In still another embodiment, the cleavage agent is in a solutioncomprising N-methyl-2-pyrrolidone.

In yet another embodiment, the deprotecting agent comprises NH₃.

In still another embodiment, the deprotecting agent is in an aqueoussolution.

In yet another embodiment, the support-medium comprises polystyrene with1% crosslinked divinylbenzene.

In another embodiment, the compound of Formula (A4) is of Formula (A4a):

wherein:

R³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and

R⁴ is selected from:

In another embodiment, the compound of Formula (A5) is of Formula (A5a):

wherein:

R¹ is a support-medium

R³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and

R⁴ is selected from:

In yet another embodiment, the compound of Formula (A8) is of Formula(A8a):

wherein:

R³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and

R⁴ is, independently at each occurrence of the compound of Formula(A8a), selected from the group consisting of:

In still another embodiment, the compound of formula (A9) is of Formula(A9a):

wherein:

n is an integer from 10 to 40,

R¹ is a support-medium,

R³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and

R⁴ is, independently for each occurrence, selected from the groupconsisting of:

In another embodiment, the compound of Formula (A10) is of Formula(A10a):

wherein:

n is an integer from 10 to 40,

R¹ is a support-medium, and

R⁴ is, independently for each occurrence, selected from the groupconsisting of:

In another embodiment, the compound of Formula (A11) is of Formula(A11a):

wherein:

n is an integer from 10 to 40, and

R⁴ is, independently for each occurrence, selected from the groupconsisting of:

In an embodiment of the oligomeric compound of Formula (A), n is 30, andR² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 G 2 T 3 T 4 G 5 C 6 C 7 T 8 C 9 C 10 G 11 G12 T 13 T 14 C 15 T 16 G 17 A 18 A 19 G 20 G 21 T 22 G 23 T 24 T 25 Cwherein the oligomeric compound of Formula (A) is a compound of Formula(G):

or a pharmaceutically acceptable salt thereof.

Golodirsen, formerly known by its code name “SPR-4053,” is a PMO havingthe base sequence 5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′(SEQ ID NO:1).Golodirsen is registered under CAS Registry Number 1422959-91-8.Chemical names include:

all-P-ambo-[P,2′,3′-trideoxy-P-(dimethylamino)-2′,3′-imino-2′,3′-seco](2′a→5′)(G-T-T-G-C-C-T-C-C-G-G-T-T-C-T-G-A-A-G-G-T-G-T-T-C)5′-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate]

Golodirsen has the following structure:

and also is represented by the following chemical structure:

Golodirsen can also be depicted by the structure of Formula (XII):

Thus, in one embodiment of the process described above, the oligomericcompound of Formula (A) is a compound of Formula (G):

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the oligomeric compound of Formula (G) is anoligomeric compound of Formula (XII):

or a pharmaceutically acceptable salt thereof.

In still another embodiment, R³ is, at each occurrence, trityl.

Processes for Preparing Golodirsen

Provided herein are processes off preparing Golodirsen.

In one aspect, provided herein is a process for preparing an oligomericcompound of Formula (G):

wherein the process comprises the sequential steps of:(a) contacting a compound of Formula (I):

wherein R¹ is a support-medium,

with a deblocking agent to form the compound of Formula (II):

wherein R¹ is a support-medium;

(b) contacting the compound of Formula (II) with compound (B):

to form a compound of Formula (III):

wherein R¹ is a support-medium;

(c) contacting the compound of Formula (III) with a deblocking agent toform a compound of Formula (IV):

wherein R¹ is a support-medium;(d) contacting the compound of Formula (IV) with a compound of Formula(DPG):

to form a compound of Formula (V):

wherein R¹ is a support-medium;

(e) contacting the compound of Formula (V) with a deblocking agent toform a compound of Formula (VI):

wherein R¹ is a support-medium;

(f) contacting the compound of Formula (VI) with a compound of Formula(T):

to form a compound of Formula (VII):

wherein R¹ is a support-medium;

(g) performing 23 iterations of the sequential steps of:

(g1) contacting the product formed by the immediately prior step with adeblocking agent; and

(g2) contacting the compound formed by the immediately prior step with acompound of Formula (VIII):

wherein R² is, independently for each compound of Formula (VIII),selected from the group consisting of:

wherein, for each iteration from 1 to 23, R² is:

Iteration No. R² 1 T 2 DPG 3 PC 4 PC 5 T 6 PC 7 PC 8 DPG 9 DPG 10 T 11 T12 PC 13 T 14 DPG 15 PA 16 PA 17 DPG 18 DPG 19 T 20 DPG 21 T 22 T 23 PC

to form a compound of Formula (IX):

wherein R¹ is a support-medium,

wherein R² is, independently for each occurrence, selected from thegroup consisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC(h) contacting the compound of Formula (IX) with a deblocking agent toform a compound of Formula (X):

wherein R¹ is a support-medium,

wherein R² is, independently for each occurrence, selected from thegroup consisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC(i) contacting the compound of Formula (X) with a cleaving agent to forma compound of Formula (XI):

wherein R² is, independently for each occurrence, selected from thegroup consisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PCand(j) contacting the compound of Formula (XI) with a deprotecting agent toform the oligomeric compound of Formula (G).

In an embodiment, step (d), step (f), step (g2), or combinations thereoffurther comprises contacting the compound of Formula (IV), Formula (VI),or the compound formed by the immediately prior step, respectively, witha capping agent.

In certain embodiments, each of step (d), step (f) and step (g2) furthercomprises contacting the compound of Formula (IV), Formula (VI), or thecompound formed by the immediately prior step, respectively, with acapping agent.

In another embodiment, each step is performed in the presence of atleast one solvent.

In yet another embodiment, the deblocking agent used in each step is asolution comprising a halogenated acid.

In still another embodiment, the deblocking agent used in each step iscyanoacetic acid.

In another embodiment, the halogenated acid is selected from the groupconsisting of chloroacetic acid, dichloroacetic acid, trichloroaceticacid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid.

In yet another embodiment, the halogenated acid is trifluoroacetic acid.

In still another embodiment, at least one of steps (c), (e), and (g1)further comprise the step of contacting the deblocked compound of eachstep with a neutralization agent.

In another embodiment, each of steps (c), (e), and (g1) further comprisethe step of contacting the deblocked compound of each step with aneutralization agent.

In yet another embodiment, the neutralization agent is in a solutioncomprising dichloromethane and isopropyl alcohol.

In still another embodiment, the neutralization agent is a monoalkyl,dialkyl, or trialkyl amine.

In another embodiment, the neutralization agent isN,N-diisopropylethylamine.

In yet another embodiment, the deblocking agent used in each step is asolution comprising 4-cyanopyridine, dichloromethane, trifluoroaceticacid, trifluoroethanol, and water.

In still another embodiment, the capping agent is in a solutioncomprising ethylmorpholine and methylpyrrolidinone.

In another embodiment, the capping agent is an acid anhydride.

In yet another embodiment, the acid anhydride is benzoic anhydride.

In still another embodiment, the compound of Formula (VIII), compound ofFormula (DPG), and compound (F) are each, independently, in a solutioncomprising ethylmorpholine and dimethylimidazolidinone.

In another embodiment, the cleavage agent comprises dithiothreitol and1,8-diazabicyclo[5.4.0]undec-7-ene.

In yet another embodiment, the cleavage agent is in a solutioncomprising N-methyl-2-pyrrolidone.

In still another embodiment, the deprotecting agent comprises NH₃.

In another embodiment, the deprotecting agent is in an aqueous solution.

In yet another embodiment, the support-medium comprises polystyrene with1% crosslinked divinylbenzene.

In another embodiment, the compound of Formula (DPG) is of Formula(DPG1):

In another embodiment, the compound of Formula (V) is of Formula (Va):

wherein R¹ is a support-medium.

In another embodiment, the compound of Formula (T) is of Formula (T1):

In another embodiment, the compound of Formula (VII) is of Formula(VIIa):

wherein R¹ is a support-medium.

In another embodiment, the compound of Formula (VIII) is of Formula(VIIIa):

wherein R² is, independently for each compound of Formula (VIIIa),selected from the group consisting of:

In another embodiment, the compound of Formula (IX) is of Formula (IXa):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is a support-medium, and

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In another embodiment, the compound of Formula (X) is of Formula (Xa):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is a support-medium, and

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In another embodiment, the compound of Formula (XI) is of Formula (XIa):

or a pharmaceutically acceptable salt thereof, wherein:

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PCIn another embodiment, the compound of Formula (VI) is of Formula (VIa):

wherein R¹ is a support-medium.

In still another embodiment, the oligomeric compound of Formula (G) isan oligomeric compound of Formula (XII):

In another aspect, provided herein is a compound of Formula (A5):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium, R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

In some embodiments, the compound of Formula (A5) is of Formula (A5a):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium, R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

In another aspect, provided herein is a compound of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium.

In some embodiments, the compound of Formula (V) is of Formula (Va):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium.

In another aspect, provided herein is a compound of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium.

In some embodiments, the compound of Formula (VI) is of Formula (Via):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium.

In another aspect, provided herein is a compound of Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium.

In some embodiments, the compound of Formula (VII) is of Formula (VIIa):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium.

In another aspect, provided herein is a compound of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a support-medium, and

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In one embodiment, the compound of Formula (IX) is of Formula (IXa):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is a support-medium, and

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In another aspect, provided herein is a compound of Formula (A9):

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 10 to 40;

R¹ is a support-medium;

R³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl; and

R⁴ is, independently at each occurrence, selected from the groupconsisting of:

In one embodiment, the compound of Formula (A9) is of Formula (A9a):

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 10 to 40;

R¹ is a support-medium;

R³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl; and

R⁴ is, independently at each occurrence, selected from the groupconsisting of:

In another aspect, provided herein is a compound of Formula (X):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is a support-medium, and

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In one embodiment, the compound of Formula (X) is of Formula (Xa):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is a support-medium, and

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In another aspect, provided herein is a compound of Formula (A10):

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 10 to 40;

R¹ is a support-medium; and

R⁴ is, independently at each occurrence, selected from the groupconsisting of:

In one embodiment, the compound of Formula (A10) is of Formula (A10a):

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 10 to 40;

R¹ is a support-medium; and

R⁴ is, independently at each occurrence, selected from the groupconsisting of:

In another embodiment of these compounds, the support-medium comprisespolystyrene with 1% crosslinked divinylbenzene.

In another aspect, provided herein is a compound of Formula (XI):

or a pharmaceutically acceptable salt thereof, wherein:

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In one embodiment, the compound of Formula (XI) is of Formula (XIa):

or a pharmaceutically acceptable salt thereof, wherein

R² is, independently at each occurrence, selected from the groupconsisting of:

and

wherein R² is at each position from 1 to 25 and 5′ to 3′:

Position No. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10DPG 11 DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22DPG 23 T 24 T 25 PC

In another aspect, provided herein is a compound of Formula (A11):

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 10 to 40; and

R⁴ is, independently at each occurrence, selected from the groupconsisting of:

In one embodiment, the compound of Formula (A11) is of formula (A11a):

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 10 to 40; and

R⁴ is, independently at each occurrence, selected from the groupconsisting of:

Oligomers

Important properties of morpholino-based subunits include: 1) theability to be linked in an oligomeric form by stable, uncharged orpositively charged backbone linkages; 2) the ability to support anucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil,5-methyl-cytosine and hypoxanthine) such that the polymer formed canhybridize with a complementary-base target nucleic acid, includingtarget RNA; 3) the ability of the oligomer to be actively or passivelytransported into mammalian cells; and 4) the ability of the oligomer andoligomer:RNA heteroduplex to resist RNAse and RNase H degradation,respectively.

In some embodiments, the antisense oligomers contain base modificationsor substitutions. For example, certain nucleo-bases may be selected toincrease the binding affinity of the antisense oligomers describedherein. 5-methylcytosine substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C., and may be incorporatedinto the antisense oligomers described herein. In one embodiment, atleast one pyrimidine base of the oligomer comprises a 5-substitutedpyrimidine base, wherein the pyrimidine base is selected from the groupconsisting of cytosine, thymine and uracil. In one embodiment, the5-substituted pyrimidine base is 5-methylcytosine. In anotherembodiment, at least one purine base of the oligomer compriseshypoxanthine.

Morpholino-based oligomers (including antisense oligomers) are detailed,for example, in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047,5,034,506, 5,166,315, 5,185,444, 5,521,063, 5,506,337, 8,299,206, and 8,076,476, International Patent Application Publication Nos.WO/2009/064471 and WO/2012/043730, and Summerton et al. (1997, Antisenseand Nucleic Acid Drug Development, 7, 187-195), each of which are herebyincorporated by reference in their entirety.

Oligomeric compounds of the disclosure may have asymmetric centers,chiral axes, and chiral planes (as described, for example, in: E. L.Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compound., John Wiley& Sons, New York, 1994, pages 1119-1190, and March, J., Advanced OrganicChemistry, 3d. Ed., Chap. 4, John Wiley & Sons, New York (1985)), andmay occur as racemates, racemic mixtures, and as individualdiasteromers, with all possible isomers and mixtures thereof, includingoptical isomers. Oligomeric compounds of the disclosure hereinspecifically mentioned, without any indication of its stereo-chemistry,are intended to represent all possible isomers and mixtures thereof.

Specifically, without wishing to be bound by any particular theory,oligomeric compounds of the disclosure are prepared, as discussedherein, from activated morpholino subunits including such non-limitingexamples such as a compound of Formula (VIII):

wherein R² is, independently for each compound of Formula (VIII),selected from the group consisting of:

Each of the above-mentioned compounds of Formula (VIII), may beprepared, for example, from the corresponding beta-D-ribofuranosyl asdepicted below:

See Summerton et al., Antisense & Nucleic Acid Drug Dev. 7:187-195(1997). Without being bound by any particular theory, the stereochemistry of the two chiral carbons is retained under the syntheticconditions such that a number of possible stereo isomers of eachmorpholino subunit may be produced based on selection of, for example,an alpha-L-ribofuranosyl, alpha-D-ribofuranosyl, beta-L-ribofuranosyl,or beta-D-ribofuranosyl starting material.

For example, in some embodiments, a compound of Formula (VIII) of thedisclosure may be of Formula (VIIIa):

wherein R² is, independently for each compound of Formula (VIIIa),selected from the group consisting of:

Without wishing to be bound by any particular theory, incorporation of10 to 40 compounds of Formula (VIII), for example, into an oligomericcompound of the disclosure may result in numerous possible stereoisomers.

Without wishing to be bound by any particular theory, oligomericcompounds of the disclosure comprise one or more phosphorous-containingintersubunits, which create a chiral center at each phosphorus, each ofwhich is designated as either an “Sp” or “Rp” configuration asunderstood in the art. Without wishing to be bound by any particulartheory, this chirality creates stereoisomers, which have identicalchemical composition but different three-dimensional arrangement oftheir atoms.

Without wishing to be bound by any particular theory, the configurationof each phosphorous intersubunit linkage occurs randomly duringsynthesis of, for example, oligomeric compounds of the disclosure.Without wishing to be bound by any particular theory, the synthesisprocess generates an exponentially large number of stereoisomers of anoligomeric compound of the disclosure because oligomeric compounds ofthe disclosure are comprised of numerous phosphorous intersubunitlinkages—with each phosphorous intersubunit linkage having a randomchiral configuration. Specifically, without wishing to be bound by anyparticular theory, each intersubunit linkage of an additional morpholinosubunit doubles the number of stereoisomers of the product, so that aconventional preparation of an oligomeric compound of the disclosure isin fact a highly heterogeneous mixtures of 2^(N) stereoisomers, where Nrepresents the number of phosphorous intersubunit linkages.

Thus, unless otherwise indicated, all such isomers, includingdiastereomeric and enantiomeric mixtures, and pure enantiomers anddiastereomers are included such as, for example, when one or more bondsfrom one or more stereo center is indicated by “-” or “˜” or anequivalent as would be understood in the art.

Table 1 depicts various embodiments of morpholino subunits provided inthe processes described herein.

TABLE 1 Various embodiments of morpholino subunits.

EXAMPLES

Examples have been set forth below for the purpose of illustration andto describe certain specific embodiments of the disclosure. However, thescope of the claims is not to be in any way limited by the examples setforth herein. Various changes and modifications to the disclosedembodiments will be apparent to those skilled in the art and suchchanges and modifications including, without limitation, those relatingto the chemical structures, substituents, derivatives, formulations ormethods of the disclosure may be made without departing from the spiritof the disclosure and the scope of the appended claims. Definitions ofthe variables in the structures in the schemes herein are commensuratewith those of corresponding positions in the formulae presented herein.

Example 1: NCP2 Anchor Synthesis 1. Preparation of Methyl4-Fluoro-3-Nitrobenzoate (1)

To a 100 L flask was charged 12.7 kg of 4-fluoro-3-nitrobenzoic acid wasadded 40 kg of methanol and 2.82 kg concentrated sulfuric acid. Themixture was stirred at reflux (65° C.) for 36 hours. The reactionmixture was cooled to 0° C. Crystals formed at 38° C. The mixture washeld at 0° C. for 4 hrs then filtered under nitrogen. The 100 L flaskwas washed and filter cake was washed with 10 kg of methanol that hadbeen cooled to 0° C. The solid filter cake was dried on the funnel for 1hour, transferred to trays, and dried in a vacuum oven at roomtemperature to a constant weight of 13.695 kg methyl4-fluoro-3-nitrobenzoate (100% yield; HPLC 99%).

2. Preparation of 3-Nitro-4-(2-oxopropyl)benzoic Acid A. (Z)-Methyl4-(3-Hydroxy-1-Methoxy-1-Oxobut-2-en−2-yl)-3-Nitrobenzoate (2)

To a 100 L flask was charged 3.98 kg of methyl 4-fluoro-3-nitrobenzoate(1) from the previous step 9.8 kg DMF, 2.81 kg methyl acetoacetate. Themixture was stirred and cooled to 0° C. To this was added 3.66 kg DBUover about 4 hours while the temperature was maintained at or below 5°C. The mixture was stirred an additional 1 hour. To the reaction flaskwas added a solution of 8.15 kg of citric acid in 37.5 kg of purifiedwater while the reaction temperature was maintained at or below 15° C.After the addition, the reaction mixture was stirred an addition 30minutes then filtered under nitrogen. The wet filter cake was returnedto the 100 L flask along with 14.8 kg of purified water. The slurry wasstirred for 10 minutes then filtered. The wet cake was again returned tothe 100 L flask, slurried with 14.8 kg of purified water for 10 minutes,and filtered to crude (Z)-methyl4-(3-hydroxy-1-methoxy-1-oxobut-2-en−2-yl)-3-nitrobenzoate.

B. 3-Nitro-4-(2-oxopropyl)benzoic Acid

The crude (Z)-methyl4-(3-hydroxy-1-methoxy-1-oxobut-2-en−2-yl)-3-nitrobenzoate was chargedto a 100 L reaction flask under nitrogen. To this was added 14.2 kg1,4-dioxane and the stirred. To the mixture was added a solution of16.655 kg concentrated HCl and 13.33 kg purified water (6M HCl) over 2hours while the temperature of the reaction mixture was maintained below15° C. When the addition was complete, the reaction mixture was heatedat reflux (80° C.) for 24 hours, cooled to room temperature, andfiltered under nitrogen. The solid filter cake was triturated with 14.8kg of purified water, filtered, triturated again with 14.8 kg ofpurified water, and filtered. The solid was returned to the 100 L flaskwith 39.9 kg of DCM and refluxed with stirring for 1 hour. 1.5 kg ofpurified water was added to dissolve the remaining solids. The bottomorganic layer was split to a pre-warmed 72 L flask, then returned to aclean dry 100 L flask. The solution was cooled to 0° C., held for 1hour, then filtered. The solid filter cake was washed twice each with asolution of 9.8 kg DCM and 5 kg heptane, then dried on the funnel. Thesolid was transferred to trays and dried to a constant weight of 1.855kg 3-Nitro-4-(2-oxopropyl)benzoic Acid. Overall yield 42% fromcompound 1. HPLC 99.45%.

3. Preparation of N-Tritylpiperazine Succinate (NTP)

To a 72 L jacketed flask was charged under nitrogen 1.805 kgtriphenylmethyl chloride and 8.3 kg of toluene (TPC solution). Themixture was stirred until the solids dissolved. To a 100 L jacketedreaction flask was added under nitrogen 5.61 kg piperazine, 19.9 kgtoluene, and 3.72 kg methanol. The mixture was stirred and cooled to 0°C. To this was slowly added in portions the TPC solution over 4 hourswhile the reaction temperature was maintained at or below 10° C. Themixture was stirred for 1.5 hours at 10° C., then allowed to warm to 14°C. 32.6 kg of purified water was charged to the 72 L flask, thentransferred to the 100 L flask while the internal batch temperature wasmaintained at 20+/−5° C. The layers were allowed to split and the bottomaqueous layer was separated and stored. The organic layer was extractedthree times with 32 kg of purified water each, and the aqueous layerswere separated and combined with the stored aqueous solution.

The remaining organic layer was cooled to 18° C. and a solution of 847 gof succinic acid in 10.87 kg of purified water was added slowly inportions to the organic layer. The mixture was stirred for 1.75 hours at20+/−5° C. The mixture was filtered, and the solids were washed with 2kg TBME and 2 kg of acetone then dried on the funnel. The filter cakewas triturated twice with 5.7 kg each of acetone and filtered and washedwith 1 kg of acetone between triturations. The solid was dried on thefunnel, then transferred to trays and dried in a vacuum oven at roomtemperature to a constant weight of 2.32 kg of NTP. Yield 80%.

4. Preparation of(4-(2-Hydroxypropyl)-3-Nitrophenyl)(4-Tritylpiperazin−1-yl)Methanone A.Preparation of1-(2-Nitro-4(4-Tritylpiperazine-1-Carbonyl)Phenyl)Propan−2-one

To a 100 L jacketed flask was charged under nitrogen 2 kg of3-Nitro-4-(2-oxopropyl)benzoic Acid (3), 18.3 kg DCM, 1.845 kgN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl).The solution was stirred until a homogenous mixture was formed. 3.048 kgof NTP was added over 30 minutes at room temperature and stirred for 8hours. 5.44 kg of purified water was added to the reaction mixture andstirred for 30 minutes. The layers were allowed to separate and thebottom organic layer containing the product was drained and stored. Theaqueous layer was extracted twice with 5.65 kg of DCM. The combinedorganic layers were washed with a solution of 1.08 kg sodium chloride in4.08 kg purified water. The organic layers were dried over 1.068 kg ofsodium sulfate and filtered. The sodium sulfate was washed with 1.3 kgof DCM. The combined organic layers were slurried with 252 g of silicagel and filtered through a filter funnel containing a bed of 252 g ofsilica gel. The silica gel bed was washed with 2 kg of DCM. The combinedorganic layers were evaporated on a rotovap. 4.8 kg of THF was added tothe residue and then evaporated on the rotovap until 2.5 volumes of thecrude 1-(2-nitro-4(4-tritylpiperazine-1-carbonyl)phenyl)propan−2-one inTHF was reached.

B. Preparation of(4-(2-Hydroxypropyl)-3-Nitrophenyl)(4-Tritylpiperazin−1-yl)Methanone (5)

To a 100 L jacketed flask was charged under nitrogen 3600 g of 4 fromthe previous step and 9800 g THF. The stirred solution was cooled to ≤5°C. The solution was diluted with 11525 g ethanol and 194 g of sodiumborohydride was added over about 2 hours at ≤5° C. The reaction mixturewas stirred an additional 2 hours at ≤5° C. The reaction was quenchedwith a solution of about 1.1 kg ammonium chloride in about 3 kg of waterby slow addition to maintain the temperature at ≤10° C. The reactionmixture was stirred an additional 30 minutes, filtered to removeinorganics, and recharged to a 100 L jacketed flask and extracted with23 kg of DCM. The organic layer was separated and the aqueous was twicemore extracted with 4.7 kg of DCM each. The combined organic layers werewashed with a solution of about 800 g of sodium chloride in about 3 kgof water, then dried over 2.7 kg of sodium sulfate. The suspension wasfiltered and the filter cake was washed with 2 kg of DCM. The combinedfiltrates were concentrated to 2.0 volumes, diluted with about 360 g ofethyl acetate, and evaporated. The crude product was loaded onto asilica gel column of 4 kg of silica packed with DCM under nitrogen andeluted with 2.3 kg ethyl acetate in 7.2 kg of DCM. The combinedfractions were evaporated and the residue was taken up in 11.7 kg oftoluene. The toluene solution was filtered and the filter cake waswashed twice with 2 kg of toluene each. The filter cake was dried to aconstant weight of 2.275 kg of compound 5 (46% yield from compound 3)HPLC 96.99%.

5. Preparation of2,5-Dioxopyrrolidin−1-yl(1-(2-Nitro-4-(4-triphenylmethylpiperazine-1Carbonyl)Phenyl)Propan−2-yl) Carbonate (NCP2 Anchor)

To a 100 L jacketed flask was charged under nitrogen 4.3 kg of compound5 (weight adjusted based on residual toluene by ¹H NMR; all reagentshere after were scaled accordingly) and 12.7 kg pyridine. To this wascharged 3.160 kg of DSC (78.91 weight % by ¹H NMR) while the internaltemperature was maintained at ≤35° C. The reaction mixture was aged forabout 22 hours at ambience then filtered. The filter cake was washedwith 200 g of pyridine. In two batches each comprising ½ the filtratevolume, filtrate wash charged slowly to a 100 L jacketed flaskcontaining a solution of about 11 kg of citric acid in about 50 kg ofwater and stirred for 30 minutes to allow for solid precipitation. Thesolid was collected with a filter funnel, washed twice with 4.3 kg ofwater per wash, and dried on the filter funnel under vacuum.

The combined solids were charged to a 100 L jacketed flask and dissolvedin 28 kg of DCM and washed with a solution of 900 g of potassiumcarbonate in 4.3 kg of water. After 1 hour, the layers were allowed toseparate and the aqueous layer was removed. The organic layer was washedwith 10 kg of water, separated, and dried over 3.5 kg of sodium sulfate.The DCM was filtered, evaporated, and dried under vacuum to 6.16 kg ofNCP2 Anchor (114% yield).

Example 2: Anchor Loaded Resin Synthesis

To a 75 L solid phase synthesis reactor was charged about 52 L of NMPand 2600 g of aminomethyl polystyrene resin. The resin was stirred inthe NMP to swell for about 2 hours then drained. The resin was washedtwice with about 39 L DCM per wash, then twice with 39 L NeutralizationSolution per wash, then twice with 39 L of DCM per wash. The NCP2 AnchorSolution was slowly added to the stirring resin solution, stirred for 24hours at room temperature, and drained. The resin was washed four timeswith 39 L of NMP per wash, and six times with 39 L of DCM per wash. Theresin was treated and stirred with ½ the DEDC Capping Solution for 30minutes, drained, and was treated and stirred with the 2^(nd) ½ of theDEDC Capping Solution for 30 minutes and drained. The resin was washedsix times with 39 L of DCM per wash then dried in an oven to constantweight of 3573.71 g of Anchor Loaded Resin.

Example 3: Preparation of Activated EG3 Tail 1. Preparation of TritylPiperazine Phenyl Carbamate 35

To a cooled suspension of NTP in dichloromethane (6 mL/g NTP) was addeda solution of potassium carbonate (3.2 eq) in water (4 mL/g potassiumcarbonate). To this two-phase mixture was slowly added a solution ofphenyl chloroformate (1.03 eq) in dichloromethane (2 g/g phenylchloroformate). The reaction mixture was warmed to 20° C. Upon reactioncompletion (1-2 hr), the layers were separated. The organic layer waswashed with water, and dried over anhydrous potassium carbonate. Theproduct 35 was isolated by crystallization from acetonitrile. Yield=80%

2. Preparation of Carbamate Alcohol (36)

Sodium hydride (1.2 eq) was suspended in 1-methyl-2-pyrrolidinone (32mL/g sodium hydride). To this suspension were added triethylene glycol(10.0 eq) and compound 35 (1.0 eq). The resulting slurry was heated to95° C. Upon reaction completion (1-2 hr), the mixture was cooled to 20°C. To this mixture was added 30% dichloromethane/methyl tert-butyl ether(v:v) and water. The product-containing organic layer was washedsuccessively with aqueous NaOH, aqueous succinic acid, and saturatedaqueous sodium chloride. The product 36 was isolated by crystallizationfrom dichloromethane/methyl tert-butyl ether/heptane. Yield=90%.

3. Preparation of EG3 Tail Acid (37)

To a solution of compound 36 in tetrahydrofuran (7 mL/g 36) was addedsuccinic anhydride (2.0 eq) and DMAP (0.5 eq). The mixture was heated to50° C. Upon reaction completion (5 hr), the mixture was cooled to 20° C.and adjusted to pH 8.5 with aqueous NaHCO₃. Methyl tert-butyl ether wasadded, and the product was extracted into the aqueous layer.Dichloromethane was added, and the mixture was adjusted to pH 3 withaqueous citric acid. The product-containing organic layer was washedwith a mixture of pH=3 citrate buffer and saturated aqueous sodiumchloride. This dichloromethane solution of 37 was used without isolationin the preparation of compound 38.

4. Preparation of Activated EG3 Tail (38)

To the solution of compound 37 was addedN-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq),4-dimethylaminopyridine (DMAP) (0.34 eq), and then1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.1eq). The mixture was heated to 55° C. Upon reaction completion (4-5 hr),the mixture was cooled to 20° C. and washed successively with 1:1 0.2 Mcitric acid/brine and brine. The dichloromethane solution underwentsolvent exchange to acetone and then to N,N-dimethylformamide, and theproduct was isolated by precipitation from acetone/N,N-dimethylformamideinto saturated aqueous sodium chloride. The crude product was reslurriedseveral times in water to remove residual N,N-dimethylformamide andsalts. Yield=70% of Activated EG3 Tail 38 from compound 36.

Example 4: 50 L Solid-Phase Synthesis of Golodirsen [Oligomeric Compound(XII)] Crude Drug Substance 1. Materials

TABLE 2 Starting Materials Material Chemical Molecular Name ChemicalName CAS Number Formula Weight Activated Phosphoramidochloridic acid,1155373-30-0 C₃₈H₃₇ClN₇O₄P 722.2 A Subunit N,N-dimethyl-,[6-[6-(benzoylamino)-9H-purin-9-yl]- 4-(triphenylmethyl)-2- morpholinyl]methylester Activated Phosphoramidochloridic acid, 1155373-31-1 C₃₇H₃₇ClN₅O₅P698.2 C Subunit N,N-dimethyl-,[6-[4- (benzoylamino)-2-oxo-1(2H)-pyrimidinyl]-4- (triphenylmethyl)-2- morpholinyl]methyl ester ActivatedPropanoic Acid, 2,2-dimethyl-, 1155309-89-9 C₅₁H₅₃ClN₇O₇p 942.2 DPGSubunit 4-[[[9-[6-[[[chloro(dimethyl- amino)phosphinyl]oxy]methyl]-4-(triphenylmethyl)-2- morpholinyl]-2-[(2- phenylacetyl)amino]-9H-purin-6-yl]oxy]methyl]phenyl ester Activated Phosphoramidochloridic acid,1155373-34-4 C₃₁H₃₄ClN₄O₅p 609.1 T SubunitN,N-dimethyl-,[6-(3,4-dihydro- 5-methyl-2,4-dioxo-1(2H)-pyrimidinyl)]-4- (triphenylmethyl)-2- morpholinyl]methyl ester ActivatedButanedioic acid, 1- 1380600-06-5 C₄₃H₄₇N₃O₁₀ 765.9 EG3 Tail[3aR,4S,7R,7aS)-1,3,3a,4,7,7a- hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl] 4- [2-[2-[2-[[[4-(triphenylmethyl)-1-piperazinyl]carbonyl]oxy]eth- oxy]ethoxy]ethyl] ester

Chemical Structures of Starting Materials: A. Activated EG3 Tail

B. Activated C Subunit (for Preparation, See U.S. Pat. No. 8,067,571)

C. Activated a Subunit (for Preparation, See U.S. Pat. No. 8,067,571)

D. Activated DPG Subunit (for Preparation, See WO 2009/064471)

E. Activated T Subunit (for Preparation, See WO 2013/082551)

F. Anchor Loaded Resin

wherein R¹ is a support-medium.

TABLE 3 Description of Solutions for Solid Phase Oligomer Synthesis ofGolodirsen Crude Drug Substance Solution Name Solution Composition NCP2Anchor 37.5 L NMP and 1292 g NCP2 Anchor Solution DEDC Capping 4.16 LDiethyl Dicarbonate (DEDC), 3.64 L NEM, Solution and 33.8 L DCM CYTFA2.02 kg 4-cyanopyridine, 158 L DCM, 1.42 L TFA, Solution 39 L TFE, and 2L purified water Neutralization 35.3 L IPA, 7.5 L DIPEA, and 106.5 L DCMSolution Cleavage 1,530.04 g DTT, 6.96 L NMP, and 2.98 L DBU Solution

2. Synthesis of Golodirsen Crude Drug Substance

A. Resin swelling

750 g of Anchor Loaded Resin and 10.5 L of NMP were charged to a 50 Lsilanized reactor and stirred for 3 hours. The NMP was drained and theAnchor Loaded Resin was washed twice with 5.5 L each of DCM and twicewith 5.5 L each of 30% TFE/DCM.

B. Cycle 0: EG3 Tail Coupling

The Anchor Loaded Resin was washed three times with 5.5 L each of 30%TFE/DCM and drained, washed with 5.5 L of CYFTA solution for 15 minutesand drained, and again washed with 5.5 L of CYTFA Solution for 15minutes without draining to which 122 mL of 1:1 NEM/DCM was charged andthe suspension stirred for 2 minutes and drained. The resin was washedonce with 5.5 L Neutralization Solution for 10 minutes and drained,twice with 5.5 L of Neutralization Solution for 5 minutes and drained,then twice with 5.5 L each of DCM and drained. A solution of 706.2 g ofactivated EG3 Tail (MW 765.85) and 234 mL of NEM in 3 L of DMI wascharged to the resin and stirred for 3 hours at RT and drained. Theresin was washed once with 5.5 L of Neutralization Solution for 10minutes and drained, once with 5.5 L of Neutralization Solution for 5minutes and drained, and once with 5.5 L of DCM and drained. A solutionof 374.8 g of benzoic anhydride and 195 mL NEM in 2680 mL NMP wascharged and stirred for 15 minutes and drained. The resin was once with5.5 L of Neutralization Solution for 10 minutes and drained, once with5.5 L of Neutralization Solution for 5 minutes and drained, and oncewith 5.5 L of DCM and drained and twice with 5.5 L each of 30% TFE/DCM.The resin was suspended in 5.5 L of 30% TFE/DCM and held for 14 hours.

C. Subunit Coupling Cycles 1-30

i. Pre-Coupling Treatments

Prior to each coupling cycle as described in FIG. 1, the resin was: 1)washed with 30% TFE/DCM; 2) a) treated with CYTFA Solution 15 minutesand drained, and b) treated with CYTFA solution for 15 minutes to whichwas added 1:1 NEM/DCM, stirred, and drained; 3) stirred three times withNeutralization Solution; and 4) washed twice with DCM. See Table 4.

ii. Post Coupling Treatments

After each subunit solution was drained as described in Table 4, theresin was: 1) washed with DCM; and 2) washed three times with 30%TFE/DCM. If the resin was held for a time period prior to the nextcoupling cycle, the third TFE/DCM wash was not drained and the resin wasretained in said TFE/DCM wash solution. See Table 4.

iii. Activated Subunit Coupling Cycles

The coupling cycles were performed as described in Table 4.

iv. Final IPA Washing

After the final coupling step was performed as described in Table 4, theresin was washed 8 times with 19.5 L each of IPA, and dried under vacuumat room temperature for about 63.5 hours to a dried weight of 4857.9 g.

D. Cleavage

The above resin bound Eteplisen Crude Drug Substance was divided intotwo lots, each lot was treated as follows. A 1619.3 g lot of resinwas: 1) stirred with 10 L of NMP for 2 hrs, then the NMP was drained; 2)washed tree times with 10 L each of 30% TFE/DCM; 3) treated with 10 LCYTFA Solution for 15 minutes; and 4) 10 L of CYTFA Solution for 15minutes to which 130 ml 1:1 NEM/DCM was then added and stirred for 2minutes and drained. The resin was treated three times with 10 L each ofNeutralization Solution, washed six times with 10 L of DCM, and eighttimes with 10 L each of NMP. The resin was treated with a CleavingSolution of 1530.4 g DTT and 2980 DBU in 6.96 L NMP for 2 hours todetach the Eteplisen Crude Drug Substance from the resin. The CleavingSolution was drained and retained in a separate vessel. The reactor andresin were washed with 4.97 L of NMP which was combined with theCleaving Solution.

TABLE 4 Pre-coupling Treatment Coupling Cycle Post-Coupling Treatment 1Quantity RT 2 30% 2 3 4 SU (g) Coupling 1 30% Cycle No.: TFE/DCM CYTFANeutralization DCM NEM (L) Time DCM TFE/DCM Subunit (SU) Wash Solution¹Solution Wash DMI (L) (Hrs.) Wash Wash  1: DPG 5.5 L a) 5.5 L 3 × 5.5 L5.5 L 536.7 g; 5 5.5 L 3 × 5.5 L b) 5.5 L, 195 ml NEM; 122 ml 3.2 L DMI 2: T 7.0 L a) 7 L 3 × 7 L 2 × 7 L 468.2 g and 4.25 7 L 3 × 7 L b) 7 L,195 ml NEM 158 ml 3.2 L DMI  3: T 8 L a) 8 L 3 × 8 L 2 × 8 L 536.7 g;4.25 8 L 3 × 8 L b) 8 L, 195 ml NEM; 182 ml 3.4 L DMI  4: DPG 9 L a) 9 L3 × 9 L 2 × 9 L 536.7 g; 4.25 9 L 3 × 9 L b) 9 L, 195 ml NEM; 206 ml 3.6L DMI  5: C 9.5 L a) 9.5 L 3 × 9.5 L 2 × 9.5 L 555.2 g; 4.25 9.5 L 3 ×9.5 L b) 9.5 L, 195 ml NEM; 220 ml 3.4 L DMI  6: C 10 L a) 10 L 3 × 10 L2 × 10 L 555.2 g; 4.25 10 L 3 × 10 L b) 10 L, 195 ml NEM; 232 ml 3.45 LDMI  7: T 11 L a) 11 L 3 × 11 L 2 × 11 L 536.7 g; 4.25 11 L 3 × 11 L b)11 L, 195 ml NEM; 256 ml 3.57 L DMI  8: C 11 L a) 11 L 3 × 11 L 2 × 11 L555.2 g; 4.25 11 L 3 × 11 L b) 11 L, 195 ml NEM; 256 ml 3.64 L DMI  9: C11.5 L a) 11.5 L 3 × 11.5 L 2 × 11.5 L 468.2 g; 4.25 11.5 L 3 × 11.5 Lb) 11.5 L 195 ml NEM; 268 ml 3.72 L DMI 10: DPG 12 L a) 12 L 3 × 12 L 2× 12 L 536.7 g; 4.25 12 L 3 × 12 L b) 12 L, 195 ml NEM; 280 ml 3.96 LDMI 11: DPG 13.5 L a) 13.5 L 3 × 13.5 L 2 × 13.5 L 721.7 g; 4.25 13.5 L3 × 13.5 L b) 13.5 L, 253 ml NEM; 204 ml 4.02 L DMI 12: T 13.5 L a) 13.5L 3 × 13.5 L 2 × 13.5 L 721.7 g; 4.25 13.5 L 3 × 13.5 L b) 13.5 L, 253ml NEM; 204 ml 4.02 L DMI 13: T 14 L a) 14 L 3 × 14 L 2 × 14 L 941.9 g;4.25 14 L 3 × 14 L b) 14 L, 253 ml NEM; 216 ml 4.02 L DMI 14: C 14.5 La) 14.5 L 3 × 14.5 L 2 × 14.5 L 941.9 g; 4.25 14.5 L 3 × 14.5 L b) 14.5L, 253 ml NEM; 228 ml 4.1 L DMI 15: T 15.5 L a) 15.5 L 3 × 15.5 L 2 ×15.5 L 721.7 g; 4.25 15.5 L 3 × 15.5 L b) 15.5 L, 253 ml NEM; 254 ml4.26 L DMI 16: DPG 15.5 L a) 15.5 L 3 × 15.5 L 2 × 15.5 L 721.7 g; 4.2515.5 L 3 × 15.5 L b) 15.5 L, 253 ml NEM; 254 ml 4.26 L DMI 17: A 16 L a)16 L 3 × 16 L 2 × 16 L 941.9 g; 4.75 16 L 3 × 16 L b) 16 L, 253 ml NEM;366 ml 4.4 L DMI 18: A 16.5 L a) 16.5 L 3 × 16.5 L 2 × 16.5 L 721.7 g;4.25 16.5 L 3 × 16.5 L b) 16.5 L, 253 ml NEM; 378 ml 4.4 L DMI 19: DPG16.5 L a) 16.5 L 3 × 16.5 L 2 × 16.5 L 608.7 g; 4.25 16.5 L 3 × 16.5 Lb) 16.5 L, 253 ml NEM; 378 ml 4.57 L DMI 20: DPG 17 L a) 17 L 3 × 17 L 2× 17 L 941.9 g; 4.75 17 L 3 × 17 L b) 17 L, 253 ml NEM; 390 ml 4.57 LDMI 21: T 17 L a) 17 L 3 × 17 L 2 × 17 L 1159.2 g; 4.25 17 L 3 × 17 L b)17 L, 311 ml NEM; 390 ml 4.72 L DMI 22: DPG 17.5 L a) 17.5 L 3 × 17.5 L2 × 17.5 L 858.7 g; 4.75 17.5 L 3 × 17.5 L b) 17.5 L, 311 ml NEM; 402 ml4.72 L DMI 23: T 17.5 L a) 17.5 L 3 × 17.5 L 2 × 17.5 L 888.3 g; 4.2517.5 L 3 × 17.5 L b) 17.5 L, 311 ml NEM; 402 ml 4.88 L DMI 24: T 18 L a)18 L 3 × 18 L 2 × 18 L 749.1 g; 4.25 18 L 3 × 18 L b) 18 L, 311 ml NEM;414 ml 4.95 L DMI 25: C 18 L a) 18 L 3 × 18 L 2 × 18 L 749.1 g; 4.25 18L 3 × 18 L b) 18 L, 311 ml NEM; 414 ml 5.1 L DMI ¹ml indicates theamount of 1:1 NEM/DCM

E. Deprotection

The combined Cleaving Solution and NMP wash were transferred to apressure vessel to which was added 39.8 L of NH₄OH (NH₃.H₂O) that hadbeen chilled to a temperature of −10° to −25° C. in a freezer. Thepressure vessel was sealed and heated to 45° C. for 16 hrs then allowedto cool to 25° C. This deprotection solution containing the Golodirsencrude drug substance was diluted 3:1 with purified water prior tosolvent removal. During solvent removal, the deprotection solution waspH adjusted to 3.0 with 2M phosphoric acid, then to pH 8.03 with NH₄OH.HPLC: C18 77.552% (FIG. 1) and SCX-10 73.768% (FIG. 2).

Example 5: Purification of Golodirsen Crude Drug Substance

The deprotection solution from Example 4, part E, containing theGolodirsen crude drug substance was loaded onto a column of ToyoPearlSuper-Q 650S anion exchange resin (Tosoh Bioscience) and eluted with agradient of 0-35% B over 17 column volume (Buffer A: 10 mM sodiumhydroxide; Buffer B: 1 M sodium chloride in 10 mM sodium hydroxide) andfractions of acceptable purity (C18 and SCX HPLC) were pooled to apurified drug product solution. HPLC: 93.571% (C18; FIG. 3) 88.270%(SCX; FIG. 4).

The purified drug substance solution was desalted and lyophilized to1450.72 g purified Golodirsen drug substance. Yield 54.56%; HPLC:93.531% (FIG. 5; C18) 88.354% (FIG. 6; SCX).

TABLE 5 Acronyms Acronym Name DBU 1,8-Diazabicycloundec-7-ene DCMDichloromethane DIPEA N,N-Diisopropylethylamine DMI1,3-Dimethyl-2-imidazolidinone DTT Dithiothreitol IPA Isopropyl alcoholMW Molecular weight NEM N-Ethylmorpholine NMP N-Methyl-2-pyrrolidone RTRoom temperature TFA 2,2,2-Trifluoroacetic acid TFE2,2,2-Trifluoroethanol

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties. Unless otherwise defined, alltechnical and scientific terms used herein are accorded the meaningcommonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

1. A process for preparing an oligomeric compound of Formula (A):

wherein n is an integer from 10 to 40, and each R² is, independently foreach occurrence, selected from the group consisting of:

wherein the process comprises the sequential steps of: (a) contacting acompound of Formula (A1):

wherein R¹ is a support-medium and R³ is selected from the groupconsisting of trityl, monomethoxytrityl, dimethoxytrityl andtrimethoxytrityl; with a deblocking agent to form the compound ofFormula (II):

wherein R¹ is a support-medium; (b) contacting the compound of Formula(II) with a compound of Formula (A2):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl; to form acompound of Formula (A3):

wherein R¹ is a support-medium, and R³ is selected from the groupconsisting of trityl, monomethoxytrityl, dimethoxytrityl andtrimethoxytrityl; (c) contacting the compound of Formula (A3) with adeblocking agent to form a compound of Formula (IV):

wherein R¹ is a support-medium; (d) contacting the compound of Formula(IV) with a compound of Formula (A4):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from the group consisting of:

to form a compound of Formula (A5):

wherein R¹ is a support-medium, R³ is selected from the group consistingof trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, andR⁴ is selected from:

(e) performing n−1 iterations of the sequential steps of: (e1)contacting the product formed by the immediately prior step with adeblocking agent; and (e2) contacting the compound formed by theimmediately prior step with a compound of Formula (A8):

wherein R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ is,independently for each compound of Formula (A8), selected from the groupconsisting of:

to form a compound of Formula (A9):

wherein n is an integer from 10 to 40, R¹ is a support-medium, R³ isselected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

(f) contacting the compound of Formula (A9) with a deblocking agent toform a compound of Formula (A10):

wherein n is an integer from 10 to 40, R¹ is a support-medium, and R⁴is, independently for each occurrence, selected from the groupconsisting of:

(g) contacting the compound of Formula (A10) with a cleaving agent toform a compound of Formula (A11):

wherein n is an integer from 10 to 40, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:

and (g) contacting the compound of Formula (A11) with a deprotectingagent to form the oligomeric compound of Formula (A).
 2. The process ofclaim 1, wherein the compound of Formula (A4) is of Formula (A4a):

wherein: R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ isselected from:


3. The process of claim 1 or 2, wherein the compound of Formula (A5) isof Formula (A5a):

wherein: R¹ is a support-medium R³ is selected from the group consistingof trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, andR⁴ is selected from:


4. The process of any one of claims 1-3, wherein the compound of Formula(A8) is of Formula (A8a):

wherein: R³ is selected from the group consisting of trityl,monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R⁴ is,independently at each occurrence of the compound of Formula (A8a),selected from the group consisting of:


5. The process of any one of claims 1-4, wherein the compound of formula(A9) is of Formula (A9a):

wherein: n is an integer from 10 to 40, R¹ is a support-medium R³ isselected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl, and R⁴ is, independently for eachoccurrence, selected from the group consisting of:


6. The process of any one of claims 1-5, wherein the compound of Formula(A10) is of Formula (A10a):

wherein: n is an integer from 10 to 40, R¹ is a support-medium, and R⁴is, independently for each occurrence, selected from the groupconsisting of:


7. The process of any one of claims 1-6, wherein the compound of Formula(A11) is of Formula (A11a):

wherein: n is an integer from 10 to 40, and R⁴ is, independently foreach occurrence, selected from the group consisting of:


8. The process of any one of claims 1-7, wherein for the oligomericcompound of Formula (A), n is 25, and R² is at each position from 1 to25 and 5′ to 3′: Position No. 5′ to 3′ R² 1 G 2 T 3 T 4 G 5 C 6 C 7 T 8C 9 C 10 G 11 G 12 T 13 T 14 C 15 T 16 G 17 A 18 A 19 G 20 G 21 T 22 G23 T 24 T 25 C

wherein the oligomeric compound of Formula (A) is a compound of Formula(G):

or a pharmaceutically acceptable salt thereof.
 9. The process of any oneof claims 1-8, wherein the oligomeric compound of Formula (G) is anoligomeric compound of Formula (XII):

or a pharmaceutically acceptable salt thereof.
 10. The process of anyone of claims 1-9, wherein R³ is, at each occurrence, trityl.
 11. Aprocess for preparing an oligomeric compound of Formula (G):

wherein the process comprises the sequential steps of: (a) contacting acompound of Formula (I):

wherein R¹ is a support-medium, with a deblocking agent to form thecompound of Formula (II):

wherein R¹ is a support-medium; (b) contacting the compound of Formula(II) with compound (B):

to form a compound of Formula (III):

wherein R¹ is a support-medium; (c) contacting the compound of Formula(III) with a deblocking agent to form a compound of Formula (IV):

wherein R¹ is a support-medium; (d) contacting the compound of Formula(IV) with a compound of Formula (DPG):

to form a compound of Formula (V):

wherein R¹ is a support-medium; (e) contacting the compound of Formula(V) with a deblocking agent to form a compound of Formula (VI):

wherein R¹ is a support-medium; (f) contacting the compound of Formula(VI) with compound of Formula (T):

to form a compound of Formula (VII):

wherein R¹ is a support-medium; (g) performing 23 iterations of thesequential steps of: (g1) contacting the product formed by theimmediately prior step with a deblocking agent; and (g2) contacting thecompound formed by the immediately prior step with a compound of Formula(VIII):

wherein R² is, independently for each compound of Formula (VIII),selected from the group consisting of:

wherein, for each iteration from 1 to 23, R² is: Iteration No. R² 1 T 2DPG 3 PC 4 PC 5 T 6 PC 7 PC 8 DPG 9 DPG 10 T 11 T 12 PC 13 T 14 DPG 15PA 16 PA 17 DPG 18 DPG 19 T 20 DPG 21 T 22 T 23 PC

to form a compound of Formula (IX):

wherein R¹ is a support-medium, wherein R² is, independently for eachoccurrence, selected from the group consisting of:

 and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC

(h) contacting the compound of Formula (IX) with a deblocking agent toform a compound of Formula (X):

wherein R¹ is a support-medium, wherein R² is, independently for eachoccurrence, selected from the group consisting of:

 and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC

(i) contacting the compound of Formula (X) with a cleaving agent to forma compound of Formula (XI):

wherein R² is, independently for each occurrence, selected from thegroup consisting of:

 and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC

and (j) contacting the compound of Formula (XI) with a deprotectingagent to form the oligomeric compound of Formula (G).
 12. The process ofany one of claims 1-11, wherein step (d), step (f) or step (g2) furthercomprises contacting the compound of Formula (IV), Formula (VI), or thecompound formed by the immediately prior step, respectively, with acapping agent.
 13. The process of any one of claims 1-12, wherein thedeblocking agent used in each step is cyanoacetic acid.
 14. The processof claim 13, wherein the halogenated acid is selected from the groupconsisting of chloroacetic acid, dichloroacetic acid, trichloroaceticacid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid.15. The process of any one of claims 1-14, wherein the support-mediumcomprises a material selected from the group consisting of glass,modified or functionalized glass, plastics (including acrylics,polystyrene (e.g., polystyrene with 1% crosslinked divinylbenzene),copolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, and TEFLON), polysaccharides, nylon ornitrocellulose, ceramics, resins, silica or silica-based materials(including silicon and modified silicon), carbon, metals, and opticalfiber bundles.
 16. The process of any one of claims 1-15, wherein theoligomeric compound of Formula (G) is an oligomeric compound of Formula(XII):


17. A compound of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is asupport-medium, and R² is, independently at each occurrence, selectedfrom the group consisting of:

and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC


18. The compound of claim 17, wherein the compound of Formula (IX) is ofFormula (IXa):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium, and R² is, independently at each occurrence, selectedfrom the group consisting of:

and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC


19. A compound of Formula (X):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium, and R² is, independently at each occurrence, selectedfrom the group consisting of:

and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC


20. The compound of claim 19, wherein the compound of Formula (X) is ofFormula (Xa):

or a pharmaceutically acceptable salt thereof, wherein R¹ is asupport-medium, and R² is, independently at each occurrence, selectedfrom the group consisting of:

and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC


21. The compound according to any one of claims 17-20, wherein thesupport-medium comprises polystyrene with 1% crosslinked divinylbenzene.22. A compound of Formula (XI):

or a pharmaceutically acceptable salt thereof, wherein: R² is,independently at each occurrence, selected from the group consisting of:

and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC


23. The compound of claim 22, wherein the compound of Formula (XI) is ofFormula (XIa):

or a pharmaceutically acceptable salt thereof, wherein R² is,independently at each occurrence, selected from the group consisting of:

and wherein R² is at each position from 1 to 25 and 5′ to 3′: PositionNo. 5′ to 3′ R² 1 DPG 2 T 3 T 4 DPG 5 PC 6 PC 7 T 8 PC 9 PC 10 DPG 11DPG 12 T 13 T 14 PC 15 T 16 DPG 17 PA 18 PA 19 DPG 20 DPG 21 T 22 DPG 23T 24 T 25 PC